Human skin comprises two compartments, namely a superficial compartment, the epidermis, and a deep compartment, the dermis.
The epidermis is composed mainly of three types of cells, which are the keratinocytes (predominant), the melanocytes and the Langerhans cells. Each of these cell types contributes by means of its intrinsic functions to the essential role played in the body by the skin, in particular the role of protecting the body against outside attacks. The dermis provides the epidermis with a solid support. It is also its nourishing element. It is mainly constituted of fibroblasts and an extracellular matrix, itself composed mainly of collagen, elastin and a ground substance. Leukocytes, mast cells and tissue macrophages are also found therein. Finally, blood vessels and nerve fibres traverse the dermis.
The skin constitutes a barrier against outside attacks, in particular: chemical, mechanical and infectious attacks, and in this respect, a certain number of defense reactions against environmental factors (climate, ultraviolet rays, tobacco, pollution, infections, etc.) and/or xenobiotic factors (such as, for example, certain medicaments) occur therein.
It is therefore essential to preserve or re-establish its integrity and the equilibrium of its various functions, in particular an equilibrium between the cell renewal and differentiation processes, or an optimum degree of moisturization.
Skin irritation is conventionally defined as a reversible and non-immunological local inflammatory reaction, characterized by an oedema and an erythema induced after simple or repeated contact between a chemical substance and the skin. Acute irritant contact dermatitis (ICD) is mainly characterized by an inflammation, whereas chronic ICD is characterized by keratinocyte hyperproliferation and transient hyperkeratosis. ICD is a multifactoral disease, the triggering of which depends on both intrinsic and extrinsic factors. Age, genetic background and sex all constitute factors which can influence the development of this pathology. Furthermore, the effects of irritants are directly linked to their chemical properties and to the concentrations applied, which influence cutaneous absorption.
Skin irritation is a very important phenomenon since it represents approximately between 60% and 80% of clinical cases of contact dermatitis. The majority of the other cases represent allergic contact dermatitis.
Substances belonging to various families of very different chemical products, such as keratin solvents, dehydrating agents or oxidizing or reducing agents may be considered to be irritants. Because of this heterogeneity, it is very difficult to propose a method for discriminating an irritant product using its chemical structure as a basis. Various irritants may induce various types of inflammations. In addition to their corrosive effects, which induce the release of preformed inflammatory mediators, chemical products may impair the cellular functions or induce the activation of the skin cells of innate immunity. This results in the release of numerous inflammation-specific compounds such as cytokines, chemokines, complement compounds and vasodilatory compounds such as histamine or the metabolites of the arachidonic acid pathway which modulate skin inflammation and cell recruitment.
Penetration of the skin by chemical compounds is a major parameter in establishing the physiopathology of ICD (Norlen L et al., J. Invest Dermatol 117: 823-829, 2001, Mizutani H et al., J. Clin Invest 87: 1066-1071, 1991). Said penetration is linked to the degree of permeability of the skin (which is linked to its physiological condition) and to the physicochemical properties of the compounds whose entry it is supposed to restrict (molecular weight, polarity, ionization stage) and the nature of the environment (excipient, carrier) through which these substances are brought into contact with the skin.
This important step corresponds to the release, from the outside environment or the carrier, of the molecule which will diffuse, and therefore to it becoming available to the body.
When an irritant comes into contact with the skin, the keratinocytes are the first cells to be activated by the chemical compound. Most of the studies on ICD have thus focused on this cell type and a large amount of data is now known about their participation in the physiopathology of ICD. Keratinocytes play an important role in the initialization of the cutaneous inflammatory reaction through the release of numerous mediators and of cytokines responsible for an entire cascade of inflammation resulting in the clinical signs of ICD. Among these, IL-1α and arachidonic acid derivatives are of particular importance in the development of the inflammation.
The release of IL-1α induces, via the activation of the NF-kB transcription factor, the transcription of genes involved in inflammation, such as the cytokines IL-1β, IL-6, GM-CSF, TNFα, chemokines, including IL-8, MCP-1, MIP-1α and eotaxin, and also the expression of adhesion molecules such as E-selectin or ICAM-1 and VCAM-1 (Gordon J R, Nature 19: 346 (6281): 274-276).
The signalling cascade generated from the activation of keratinocytes begins from the release of prestored key mediators. In fact, resting keratinocytes contain large amounts of preformed and biologically active IL-1α (Marks F et al., Toxicol Lett 96: 111-118, 1998), and also of arachidonic acid (Murphy J E et al., J Invest Dermatol 114: 602-608, 2000). Because these two compounds are constitutively produced by keratinocytes, and remain stored in the cell, the epidermis may be considered to be a major reservoir of highly inflammatory mediators. An impairment of the keratinocytes due to the corrosive effect of a chemical compound, a burn or UV exposure induces the release of IL-1α and of arachidonic acid, which become the body's first defense events. IL-1α not only has an autocrine role, but has been described as inducing the transcription of more than 90 different genes on various skin cell types, such as keratinocytes, endothelial cells or fibroblasts, through the activation of the NF-kB transcription factor pathway (Gordon J R, Nature 19: 346 (6281): 274-276).
Arachidonic acid is, for its part, rapidly metabolized to many highly active compounds, eicosanoids such as prostaglandins, thromboxane and leukotrienes acting as local mediators with a short lifetime, involved in the control of proliferation, differentiation, apoptosis, or else the formation of oedema or leukocyte activation (Murphy J E et al., J Invest Dermatol 114: 602-608, 2000).
Thus, IL-1α and arachidonic acid could be considered to be the key mediators for triggering the irritation in response to a chemical stress (Murphy J E et al., J Invest Dermatol 114: 602-608, 2000).
Among all the inflammatory mediators, besides IL-1s and arachidonic acid, only TNF-α can activate a sufficient number of mechanisms to independently generate a skin inflammation. This major cytokine of skin inflammation is already prestored in the dermal mast cells (Larrick J W et al., J Leukoc Biol 45: 429-433, 1989) but is also produced by the keratinocytes and the Langerhans cells after stimulation (Groves R W, et al., J Invest Dermatol 98: 384-387, 1992). One of the mechanisms through which TNF-α has most influence on the inflammatory reaction is the induction of adhesion molecules in synergy with IL-1. Adhesion molecules play an essential role in the circulation and penetration of leukocytes (in particular neutrophils) from the peripheral blood vessels to the dermis and the epidermis (Holliday M R et al., Am J Contact Dermat 8: 158-164, 1997).
A large amount of chemical products can induce a skin irritation; however, they differ in terms of their ability to generate pro-inflammatory cytokines and a skin inflammation is not systematically dependent on the production of TNF-α.
It is important to also note that the production of IL-12 and of IL-18 by the activated macrophages at the site of the inflammation plays an important role as a local amplification loop. This is because these cytokines stimulate the production of IFN-γ by neighbouring T lymphocytes, which is in turn a powerful coactivation factor for the macrophages and the keratinocytes.
Furthermore, chemokine secretion by the vascular cells and the keratinocytes occurs. The chemokines of the CC subgroup (so-named since the first cysteines are contiguous), the prototype of which is IL-8, are chemotactic with respect to polymorphonuclear cells and certain lymphocytes. The chemokines of the CXC group (so-named because an amino acid is integrated between the first two cysteines), the prototype of which is MCP-1 (monocyte chemoattractant protein 1), are chemotactic with respect to monocytes/macrophages and certain lymphocytes. These two types of chemokines released at the inflammation site explain the polymorphic cellular infiltrate found in an inflamed tissue.
IL-6 is also a cytokine secreted by keratinocytes, macrophages and vascular cells activated during an inflammation. This important cytokine is involved in numerous systems, such as immune response, haematopoiesis, osteoblast proliferation, etc. In skin irritations, IL-6 is produced locally and may reach the general circulation and trigger regional and general effects.
In parallel to the cytokines and chemokines and also the arachidonic acid metabolites, another major inflammation mediator is oxidative stress.
Certain chemical irritants are known to generate free radicals and ROSs (reactive oxygen species) capable of inducing lipid peroxidation or DNA alteration. The hypothesis that oxidative stress plays a role in the phenomena of irritation induced by a chemical compound is supported by the fact that ROS inhibitors/scavengers inhibit skin inflammation (Zhang L et al., J Invest Dermatol 115: 168-176, 2000).
Although Langerhans cells (LC) play a fundamental role in the induction of an antigen-specific response, they do not appear to have a major role in the physiopathology of ICD. Many studies have described changes in morphology or in density of LCs after epicutaneous application of irritants (Mikulowska A et al., Contact Dermatitis 34: 397-401, 1996; Kimber I et al., J Invest Dermatol 99: 48S-50S, 1992). However, these results could rather represent a non-specific response to the inflammatory reaction generated. It is now obvious that the IL-1α and the TNF-α produced during ICD have the ability to cause LCs to migrate in a dose-dependent manner. It may thus be that local concentrations of IL-1α and of TNF-α induced by irritants can generate a variable migration of LCs (Kimber I et al., J Invest Dermatol 99: 48S-50S, 1992).
Among the various populations of the cutaneous innate immune system, the mast cell is a major cell in the development of ICD. Mast cells are present in the dermis close to the blood vessels and are the only cells to contain prestored and biologically active TNF-α (Larrick J W et al., J Leukoc Biol 45: 429-433, 1989).
As opposed to these pro-inflammatory cytokines, there exist anti-inflammatory cytokines such as TGF-β and IL-10, and also shock proteins known to modulate any “danger” signal (i.e.: BiP (grp78) and HSP27) (Panayi G et al., Cur Opinion Immunol 16: 531-534, 2004).
It is therefore desirable to find novel ways for strengthening the skin's resistance to these deleterious mechanisms, and therefore for avoiding, treating or preventing inflammation and immunological disorders.
Application WO 02/098365 (Advanced Tissue Science) proposes the use of conditioned culture media for cosmetic or pharmaceutical applications. The conditioned media are obtained by culturing human skin cells, in particular fibroblasts or keratinocytes. These cells are genetically modified to increase their production of growth factors or of antioxidants in the medium, which generally contains a soluble collagen.
US 2005/0249691 describes cosmetic or dermatological compositions comprising a culture medium for cells of the skin or the horny layers, in combination with a gelled matrix; the compositions necessarily contain collagen, chitosans and glycosaminoglycans.
US 2006/0182701 also describes cosmetic or dermatological compositions comprising a culture medium for skin cells. It aims to provide the skin cells, to which the compositions are applied, with a medium which will allow them to develop in a similar manner to that which is obtained in vitro.